GB2243153A - Restriction enzyme ex. Brevibacterium - Google Patents
Restriction enzyme ex. Brevibacterium Download PDFInfo
- Publication number
- GB2243153A GB2243153A GB9107589A GB9107589A GB2243153A GB 2243153 A GB2243153 A GB 2243153A GB 9107589 A GB9107589 A GB 9107589A GB 9107589 A GB9107589 A GB 9107589A GB 2243153 A GB2243153 A GB 2243153A
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- Prior art keywords
- restriction enzyme
- dna
- enzyme
- producing
- nucleotide sequence
- Prior art date
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- 108091008146 restriction endonucleases Proteins 0.000 title claims abstract description 36
- 241000186146 Brevibacterium Species 0.000 title claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 17
- 244000005700 microbiome Species 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 claims description 6
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 4
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 claims description 3
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 claims description 3
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 claims description 3
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 claims description 3
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 claims description 3
- 229940104230 thymidine Drugs 0.000 claims description 3
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims description 2
- 229960005305 adenosine Drugs 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 abstract description 14
- 102000004190 Enzymes Human genes 0.000 abstract description 14
- 108020004414 DNA Proteins 0.000 description 17
- 239000007853 buffer solution Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 102000053602 DNA Human genes 0.000 description 4
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- 238000005273 aeration Methods 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 2
- 241000701109 Human adenovirus 2 Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 2
- 229940080469 phosphocellulose Drugs 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 239000008057 potassium phosphate buffer Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 235000012539 Bacterium linens Nutrition 0.000 description 1
- 241000186310 Brevibacterium linens Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241001596203 Trichormus variabilis UW Species 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 101150093826 par1 gene Proteins 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/84—Brevibacterium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/843—Corynebacterium
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
A method of producing a restriction enzyme capable of recognising and cleaving the same DNA base sequence as Avr II by cultivating a microorganism belonging to the genus Brevibacterium, from which the enzyme is isolated, is described.
Description
NEW RESTRICTION ENZYME This invention relates to a restriction enzyme.
More part i c u 1 a r l y, it relates to a process for producing a restriction enzyme by the cultivation of a microorganism '.jelongiii(j. to the genus Brevibacterium.
Restriction enzymes are endonucleases that are capable of recognizing a specific nucleotide sequence of in a deoxy ribonucleic acid ( DNA) molecule and cleaving the double strande,i DNA at specific sites. Pis a result of the jrogress in the molecular genetics, biochemistry and related sciences, DNA proved to hold the key to the hereditary constitution of living bodies, and since then restriction enzymes have been extensively used as useful enzymes for various purposes, such as clarification of genetic diseases and mass- production of useful substances by genetic wani pu la t i on. Restriction enzymes have been isolated fron a variety of microorganisms, and about 150 kinds are known at present, each being identified Iny the specific nucleotide sequence it recognizes an(] by the cleavage pattern it exhibits, As a restriction enzyme capable of recognizing the following nucleotide sequence and cleaving it at the arrowmarked sites:
5'- CC T A G G - 3'- G G A T Cf - ( wherein C, T, A and G represent cytidine, thymidine, j- a'(leilosinf-. and guanosine, respectively), is known Avr II produced by Anabaena variabilis UW [ Gene, Vol. 7, p.217-270.1979) 1.
This Avr II-producing microorganism is an alga, which is difficult to cultivate and produces Avr II in a very low yield; lience, the use of this microorganism is not amenable to industrial production.
Ihe object of this invention is to provide a metliod of producing a restriction enzyme capable of recognizing and cleaving the same base sequence as Avr II, which is amenable to industrial production.
Lriefly, this invention relates to a process for producing a restriction enzyme, which comprises growing a microorganism belonging to the genus Brevibacterium arid capable of producing a microorganism that specifically cleaves the following necleotide sequence at the arrowmarkted sites, and recovering said restriction enzyme from the culture broth.
5'- A T A G G - 3' V- G G A T C t C - 5, wherein C, T, A and G represent cytidine, thymidine, adenosine and guanosine, respectively.
The present inventors found that a restriction enzyme 2 capable of recognizing and cleaving the same specific nucleotide set-ltie-iice as Avr IT can be produced in a large amount by tile cultivation of a microorganism belonging to the genus Brevibacterium, and that said microorganism does not produce any restriction enzyme other than Avr II and hence the enzyme formed can be easily purified. This invention was accomplished on the basis of these f indings.
This invention will be described below in more detail.
Any microorganism belonging to the genus Brevibacterium and capable of producing said restriction enzyme may be used in the process of this invention. As an example, may be mentioned Brevibacterium lines IArc11902 stored at the Applied Microorganism Laboratory in Tokyo University and desposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, under FERM BP-2870. on 16 April 1990.
The culture -medium used in tile process of this invention contains a proper combination of carbon resources, nitrogen resources, inorganic salts and other nutrients which the microorganism used will assimilate to produce the restriction enzyme. Its pH should preferably be in the range from 5.0 to 9.0. Any of shake culture, agitation culture and aeration culture may be adopted, but cultivation with aeration and agitation is preferred for massproduction. The culture temperature may be in the temperature range that ensures production of the restriction 3 enzyine, but a range from 20 to 330C is the most preferred. The optimal cultivation time varies depending on the culture conditions adopted, and cultivation should be continued until the yield of the restriction enzyme reaches its maximum.
The restriction enzyme produced by the process of this invention is chiefly accumulated inside the microbial cells, and the grown cells can be isolated from the culture broth, for example, by centrifugation.
The enzyme formed can be isolated and purified by using known techniques commonly employed for restriction enzymes. The collected microbial cells are dispersed in a buffer solution, treatment, removal of is treated a iTt in on i u m precipitate a Tris-11C1 the cell walls were broken down by ultrasonic and the accumulated enzyme is extracted. After the residue by ultracentrifugation, the extract with 1% streptomycin to remove nucleic acids, and sulfate is then added for salting out. The w1hich separates out is collected and dissolved in buffer solution ( p1l 7.5), and the solution is dialyzed against the same buffer solution. The dialyzate is then purified by ion-exchange chromatography using phosphocellulose and hydroxyapatite, or by affinity chromatography using heparine- Sepha rose, thus giving the restriction enzyme of this invention.
The activity of this enzyme was determined according to' the method decribed below. A substrate solution of the 4 composition shown in Table 2 below was prepared.
MM 7 mM 7 rrtM 100 MM 1.0 1- g Table 1 Tris-VIC1, pII 7.5 M9C12 2-Mercaptoethanol NaCl ',-DNA ( product of Takara Shuzo Co. Ltd.
This solution ( 50_,.ul) was preheated to 370C, a sample of the enzyme of this invention to be tested was then added to allow the enzymatic reaction to proceed at that temperature, and the reaction was stopped 60 minutes later by adding 5,Z(l of a terminator solution ( containing 1% SDS, 50% glycerol, and 0.02% Bromophenol Blue). The reaction i,,iixture was applied to a 1% agarose slab gel, and electropnoresis was conducted at a constant voltage of 10 V/cm for about one to two hours. The buffer solution used for electrophoresis was 90mM Tris-borate buffer containing 2.5mil EDTA ( pII 8.3). DMA bands can be detected by UV irradi- ation if 0.5 g/ml ethidium bromide is previously added to the gel. Llectrophoresis was regarded as complete when the number and intensity of the bands for DNA fragments no longer changed.
The enzyme activity which ensures complete digestion of 1 /Lg ?:-DNA after one hour's reaction at 370C was defined as one unit.
- The restriction enzyme obtained by the process of this invention has the physicochemical properties as describe-d below. (1) Action and substrate specificity This enzyme is capable of recognizing the nucleotide sequence in a double- stranded DNA molecule as shown below and cleaving it at the arrow-marked sites, and is therefore an isoschizomer of the known restriction enzyme Avr II.
5'- C C T A G G - 3' 3'- G G A T C C - S' The base sequence recognized by the restriction enzyme of this invention was determined by using, as substrate, /-\DNA, I)BR322 DNA and 6X 174RFI DNA ( products of Takara Shuzo Co., Ltd. as well as adenovirus-2 DNA ( product of Bethesda Research Laboratories). The result was that the restriction enzyme of this invention cleaved X-DNA at two sites and adenovirus-2 DNA at two sites, but failed to cleave pBR322 DNA and 6X 174RFI DNA. In addition, the known restriction enzyme, Avr II, was allowed to act upon these substrates, and the cleavage patterns thus obtained were compared with those of the restriction enzyme of this 6 A invention, demonstrating the same patterns between the two types of enzymes. These data led to the conclusion that the nucleotide sequence in DNA molecules which the restric- tlon enzyme of this invention recognizes is 5'-CCTAGG-3'.
The sites of cleavage by the restriction enzyme of this invention was determined by recovering a single-stranded DNA from a vector prepared by introducing 5'-GCCTAGGC-3' ( a nucleofide sequence including 5'-CCTAGG-3' which is the sequence recognized by the restriction enzyme of this invention) to M13 mp18 RFI DNA ( product of Takara Shuzo Co., Ltd.
annealing it with a primer of 5'-GTTTTCCCAGTCACGAC-3' labelled with 32 P at 5' end, synthesizing a double-stranded chain by the use of E. coli DtZA polymerase I Klenow fragment, cleaving the doublestranded DNA thus obtained by the restriction enzyme of this invention, and measuring the chain length of fragments thus formed by electrophoresis on a modified polyacrylamide gel. The obtained product was detected as a spot formed IDY cleavage at the arrow-marked site of 5'-ACTAGG-3', leading to the conclusion that the enzyme of this invention recognizes the following nucleotide sequence and to cleave it at the arrow-marked sites.
5'-CC TAG G-3' 3'- G G A T C C - 5' 7 (2) Optimal conditions for enzymatic activity a) Optimal temperature The optimal temperature was approximately 370C. Optimal pli The optimal pH was in the range from 7.0 to 8.5. Salt concentration The optimal salt concentration was in the range from 50 to 150mi-I in the case of NaCl.
1) i19C12 concentration The enzymatic reaction of the restriction enzyme of this invention was activated at a MgCl2 concentration in the rangefrom 5mM to 20mM.
Molecular weight The molecular weight of the restriction enzyme of this invention was 96OOOt4OOO daltons when measured by the gel filtration method using Sephadex G-100, and was 42000t2OOO aaltons when measured by electrophoresiS 01-1 SDS-polyacrylamide gel. This indicates that this enzyme is a dimeric enzyme composed of two subunits having a molecular weight of about 45000 daltons.
c) The following Example will further illustrate this invention but is not intended to limit its scope.
8 A i Example 1
T,aenty liters of a culture medium having the composition shown in Table 2 below was put in a 30-liter jar fermenter and sterilized by the method commonly employed.
Table 2 Glucose Yeast extract Polypeptone Sodium chloride Deionized water pH 1 9 5 9 10 g 5 9 1 1 7.2 Inoculum (500 ml) of Brevibacterium linens IAM 1902, (FEFM BP-2870 obtained by shake culture in a medium having the sarne composition as above at 300C for 24 hours, was placed in the ahove jar fermenter, and cultivation was conducted at 300C for 18 hours with aeration ( 1 vvm) and agitation ( 250 rpm). The grown cells were collected from the culture broth by using a refrigerated centrifuge ( about 144 grams of grown cells on wet basis from 20 liters of the culture broth).
Seventy-two grams of the microbial cells obtained above were suspended in 360 ml of buffer solution A ( containing 20mM Tris-14C1 ( pH 7.5), 10mM 2mercaptoethanol and 5% 9 glycerol), the suspension was treated in a ultrasonic crusher to break down the cell walls, and the resulting inixture was centrifuged ( 100,000 x g, one hour) to remove the residue.
To the extract thus obtained ( 400 ml), was added 4 of streptomycin, and the mixture was allowed to stand at 40C for one hour and centrifuged ( 10, 000 x g, 10 minutes). To tho supernatant thus obtained, was added ammonium sulfate to 80% saturation, the precipitate which separated out was collected by centrifugation and dissolved in buffer solution A further containing 0.2M KC1, and the solution was dialyzed overnight against the same buffer solution as above.
T Ihe dialyzate was then adsorbed on 100 ml of phosphocellulose ( product of Whatman Co.) packed in a column and previously equilibrated with buffer solution A containing 0.2:4 XC1. After washing with the same buffer as above, the;idsorbed portion was eluted with buffer solutions A containing 0.21,1 to 1.01-1 KCI ( linear concentration gradient technique The active fractions thus obtained were mixed toyether, the combined solution was then adsorbed on 30 ml of hydroxyapatite ( Bio-rad Laboratories Ltd.) packed in a column and previously equilibrated with 10mM potassium phosphate buffer solution, and the adsorbed portion was eluted with 10mM to 5OOmM potassium phosphate buffer solutions ( linear concentration gradient technique). The solution A and active fractions thus obtained were mixed solution was dialyzed for four hours the dialyzate he par 1 ne- Se pha rose ( product of Inc.) previously equilibrated After thoroughly washing with the same buffer together, the against buffer was once more adsorbed on Pharmacia Fine Chemicals with buffer solution A.
as above, the adsorbed portion was eluted with buffer solution A containing O.BM KC1, affording the standard sample of the restriction enzyme of this invention.
This standard sample was free from any nonspecific DNIase or phosphatase.
The purification method described above gave 800,000unit activity from 72 g of wet microbial cells.
As is apparent from the foregoing, this invention provides an industrially advantageous process for producing a restriction enzyme capable of recognizing and cleaving the same base sequence as Avr II.
Claims (1)
- WHAT WE CLAIM IS:. A process for producing a restriction enzyme, which c.omprises cultivating a microorganism belonging to the genus Brevibacterium and capable of producing a restriction enzyme that cleaves the following nucleotide sequence specifically at the arrow-marked sites:A 5'- C4c- T A G G - 3' 3'- G G A T C 4\ C - 5' ( wherein C, T, A and G represent cytidine, thymidine, adenosine and quanosine, respectively), and recovering from the culture broth the restriction enzyme which cleaves the above nucleotide sequence specifically at the arrow-marked sites.Published 1991 at The Patent Office. Concept House. Cardiff Road. Newport. Gwent NP9 I RH. Further copies may be obtained from Sales Branch. Unit 6. Nine Mile Point. Cwmfelinfach, Cross Keys. Newport. NP1 7HZ. Printed by Multiplex techniques lid. St Mary CraLv. Kent.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2106231A JPH066056B2 (en) | 1990-04-20 | 1990-04-20 | Method for producing restriction enzyme |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB9107589D0 GB9107589D0 (en) | 1991-05-29 |
| GB2243153A true GB2243153A (en) | 1991-10-23 |
| GB2243153B GB2243153B (en) | 1993-08-04 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB9107589A Expired - Lifetime GB2243153B (en) | 1990-04-20 | 1991-04-10 | Isoschizomer of avr ii extracted from brevibacterium sp |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US5470732A (en) |
| JP (1) | JPH066056B2 (en) |
| DE (1) | DE4112563A1 (en) |
| GB (1) | GB2243153B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58107172A (en) * | 1981-12-18 | 1983-06-25 | Ajinomoto Co Inc | Mutant |
-
1990
- 1990-04-20 JP JP2106231A patent/JPH066056B2/en not_active Expired - Fee Related
-
1991
- 1991-04-10 GB GB9107589A patent/GB2243153B/en not_active Expired - Lifetime
- 1991-04-17 DE DE4112563A patent/DE4112563A1/en active Granted
- 1991-04-22 US US07/688,426 patent/US5470732A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US5470732A (en) | 1995-11-28 |
| GB2243153B (en) | 1993-08-04 |
| JPH044875A (en) | 1992-01-09 |
| DE4112563A1 (en) | 1991-10-24 |
| JPH066056B2 (en) | 1994-01-26 |
| GB9107589D0 (en) | 1991-05-29 |
| DE4112563C2 (en) | 1992-08-20 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 732E | Amendments to the register in respect of changes of name or changes affecting rights (sect. 32/1977) | ||
| PE20 | Patent expired after termination of 20 years |
Expiry date: 20110409 |